The majority of the common solid tumors have variable but substantial hypoxic fractions. These hypoxic cells are resistant to many chemotherapeutic drugs including alkylating agents. The basis for this resistance is poorly understood. In the previous funding period we found that hypoxic exposure induces the overexpression of a number of enzymes involved in the detoxication of xenobiotics, including anticancer drugs. We found a 4-fold increase in DT diaphorase mRNA in HT29 cells after an 8- hour hypoxic exposure. mRNAs for other detoxicating enzymes were elevated to a similar degree. We propose to investigate the mechanism underlying this phenomenon by determining the basis for increased steadystate mRNA levels. We will also determine the effect of hypoxia on DT diaphorase protein content and investigate post-transcriptional mechanisms as a possible locus of control. We also focus on a novel nuclear protein named Ref-l that appears to control the affinity of various transcription factors for their promoter elements. We will investigate the possible role of this protein in mediating the hypoxic response. The long-term goal of this work is to identify how the expression of detoxicating enzymes is controlled under hypoxia to permit therapeutic intervention directed to novel targets in the future.